Colonization of Dogs and Their Owners with Staphylococcus aureus and Staphylococcus pseudintermedius in Households, Veterinary Practices, and Healthcare Facilities

There are uncertainties with respect to the transmission of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) and Staphylococcus pseudintermedius between dogs and humans. In this study, we investigated concomitant nasal colonization of dogs and humans in three cohorts. Cohort I, households owning dogs: In 42 of 84 households, 66 humans (36.9%) and 10 dogs (8.9%) carried S. aureus. MRSA, attributed to sequence type (ST) 22 and ST130, were detected in two (1.1%) of the humans but in none of the dogs. Typing by means of spa-typing and whole-genome sequencing (WGS) indicated eight transmissions of S. aureus between humans and dogs in 8 of 42 (19.0%) households with human S. aureus carriers, whereas in 11 of 38 (29.0%) households with ≥two persons and S. aureus colonization of humans, 15 human-to-human transmissions were observed (p = 0.43). S. pseudintermedius was isolated from 42 dogs (37.5%), but from only one human (0.6%). In this case, WGS-based typing indicated strong relatedness of this isolate with a canine isolate from the same household. Cohort II, dogs and their owners visiting a veterinary practice: Among 17 humans and 17 dogs attending a veterinary practice, MSSA was detected in three humans and two dogs, and S. pseudintermedius in only six dogs. Cohort III, dogs used for animal-assisted interventions in human healthcare facilities and their owners: MSSA was obtained in 1 of 59 dogs (1.7%) and in 17 of 60 (28.3%) of the dog owners, while S. pseudintermedius was isolated from seven (12%) dogs and one (1.7%) human owner. We conclude that the risk of exchanging S. aureus/MRSA between humans and dogs is higher than that for S. pseudintermedius.


Introduction
Staphylococcus aureus (S. aureus) is widely disseminated as a colonizer and as an opportunistic pathogen among humans and other animal species. In the human community, it colonizes the anterior nares of about 25-35% of healthy persons [1,2]. The population structure of S. aureus consists of several clonal complexes, some of which are associated with defined animal species, while others are less host-specific [3]. Methicillin-resistant S. aureus (MRSA) is globally prevalent in nosocomial settings as healthcare-associated MRSA (HA-MRSA), which is mainly due to the spread of epidemic clonal lineages [4]. In addition, MRSA emerges in the community without any relation to the healthcare facilities (CA-MRSA, [5]. HA-MRSAs, especially those attributed to lineage ST22, were also observed as nosocomial pathogens in veterinary hospitals [6]. Livestock-associated MRSA (LA-MRSA), attributed to clonal complex (CC) 398 as defined by multilocus sequence typing (MLST), originally emerged in livestock before a particular subpopulation became increasingly prevalent in animal hospitals during the past decade [7][8][9].

Materials and Methods
Three different cohorts of participants were enrolled: households owning dogs (cohort I), dogs and their owners visiting a veterinary practice (cohort II), and dogs used for AAI in human healthcare facilities and their owners (cohort III). The study was performed from 2019-2020.

Study Populations and Recruitment
Cohort I: The study was performed in the Landkreis Harz region in Central Germany (214,446 inhabitants, 2104.54 km 2 ). There are three primary care hospitals in this area. A total of 83 households were enrolled. Recruitment was based on directly contacting several families owning dogs in five villages and three towns. In a second step, households already participating in the study referred to other dog-owning households. A prerequisite for participation was the agreement of the contacted households, documented by the return of a declaration of consent. The participants were provided with swabs, as well as an instruction for swab-self-collection for humans and dogs, and a short questionnaire asking for information on basic demographic characteristics, such as age, sex, and potential risk factors for colonization with S. aureus (e.g., antibiotic treatment, hospital stay during the six months prior to sampling). The feasibility of self-collection of nasal swabs was previously reported [33]. Households in which dogs with S. aureus carriage were detected were asked to provide a second nasal sample from the dogs and owners 12 months after the initial sampling.
Cohort II: 17 dogs and their owners were randomly selected by a veterinarian and instructed with respect to self-sampling and data provision as in cohort I.
Cohort III: Supported by the organization "Animals as Therapy" (www.tierealstherapie. at/tat-waz/, accessed on 21 February 2022) the owners of dogs used for AAI in human regional healthcare facilities were directly contacted. The further procedure was as described for cohort I.
The study was approved by the ethical committee of the medical faculty of the University of Magdeburg (#33/14).

Microbiological Analysis
The eSwab TM system (MAST Diagnostics, Reinfeld, Germany) was used for taking swabs from both nostrils of humans and dogs. Aliquots were streaked on CHROMagar TM MRSA (MAST) and, in parallel, on Mueller-Hinton blood agar plates (Oxoid, Wesel, Germany). After incubation at 37 • C for 18-24 h, one suspicious colony was subcultured on sheep blood agar (except in the case of differences in colonial morphology and hemolysis). Confirmation of S. aureus was performed by demonstration of the clumping factor and, additionally, by the tube coagulase test. For proof of the clumping factor, we used a solution of fibrinogen from human plasma (Sigma-Aldrich, Taufkirchen, Germany) of 2 mg/mL 0.85% NaCl. For the tube coagulase test, fresh ready-to-use human plasma (DRK blood donation service, Springe, Germany) was used. In the case of negative results, we performed a PCR for the region of tuf that is specific for S. aureus using the primers and PCR conditions as described [34]. For the identification of S. pseudintermedius, presumptive colonies were subcultured on blood agar plates and subjected to PCR analysis according to [35]. For PCR analysis, genomic DNA was extracted using the DNeasy tissue kit (Qiagen, Hilden, Germany), and lysostaphin (100 mg/L; Sigma, Taufkirchen, Germany) for bacterial lysis.

Molecular Characterization
DNA extraction: Strains were grown overnight in tryptic soy broth at 37 • C. For PCR analysis, genomic DNA was extracted using the DNeasy tissue kit (Qiagen, Hilden, Germany), and lysostaphin (100 mg/L; Sigma, Taufkirchen, Germany) for bacterial lysis.
PCR for resistance genes: PCR analysis was performed for mecA and mecC in order to confirm MRSA as previously described [36].

Whole-Genome Sequencing and Phylogenetic Analyses
Whole-genome sequencing: DNA quantification was carried out using the Qubit dsDNA HS Assay Kit (Invitrogen/Thermo Fisher Scientific, Karlsruhe, Germany). A total of 1 ng of extracted DNA was employed for library preparation using the Nextera XT DNA Library Prep Kit according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Sequencing was performed on an Illumina MiSeq platform in paired-end mode with a final readout of 2 × 250 bp. The quality of the raw read data was assessed by an in-house-developed pipeline (Qcumber-2). Genome assembly and core-genome multilocus sequence typing (cgMLST): The raw read files were imported into Ridom SeqSphere+ version 8.1.0 (Ridom GmbH, Münster, Germany (https://www.ridom.de/ seqshpere/ug/v81/Version_History.html, accessed on 21 February 2022) and assembled de novo using SPAdes (version 3.13.1 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3 342519/; accessed on 21 February 2022), default settings, integrated in the Linux version of SeqSphere). For the S. aureus isolates, the S. aureus cgMLST scheme version 1.3 (http: //pubmed.ncbi.nlm.nih.gov/24759713/; accessed on 21 February 2022), implemented into SeqSphere+, was applied. For S. pseudintermedius, an ad hoc cgMLST was established according to Ridom SeqSphere guidelines, with S. pseudintermedius ED99 as a seed genome (GenBank accession number CP002478). Minimum spanning trees were calculated on the basis of cgMLST by ignoring pairwise missing values. To detect closely related S. aureus and S. pseudintermedius isolates, the threshold for cluster distance determination was set to 10 and 15 cgMLST alleles, respectively.

Results
This study included 179, 17, and 60 humans in cohorts I, II, and III, respectively. Moreover, 112, 17, and 59 dogs were included in the three cohorts.

Cohort I: Humans and Dogs in Household Contacts
Overall, 179 persons participated in the study, representing all members of 84 households. Among these households, 18 were single households (21.4%), 48 comprised two persons (45.2%), and 18 comprised ≥ three persons. In the 84 households, there were 112 dogs, which were included in the study (one dog in 61 households, two dogs in 19 households, ≥three dogs in 4 households).
3.1.1. S. aureus Nasal Carriage S. aureus was detected in 66/179 human participants (36.9%); 64 (35.8%) carried methicillin-susceptible S. aureus (MSSA), and 2 carried MRSA (1.1%). S. aureus nasal carriage was significantly more frequent among persons suffering from atopic eczema ( Table 1). For the other variables, an association with S. aureus carriage was not apparent. For all 112 dogs living in these households, the overall distribution of risk factors for S. aureus carriage was as follows: a previous stay in an animal hospital was reported for 8 (6.6%), a previous antibiotic prescription for 25 (21.5%), skin lesions for 6 (5.4%), and bite injuries for 1 (0.9%). Among these 112 dogs, 10 (7.8%) carried S. aureus (all of which were MSSA). Of these, two were previously treated with an antibiotic, one was treated in a veterinary hospital, and one had a skin lesion.
Typing attributed the S. aureus isolates of humans to MLST clonal complexes CC1 (3), CC5 (1), CC7 (4), CC8 (9), CC12 (2), CC15 (12), CC22 (5), CC30 (10), CC45 (15), CC97 (1), CC398 (1), CC130 (1), and ST133 (3). The two MRSA isolates were associated with CC22 (harboring mecA; phenotypically resistant to penicillin, cefoxitin, oxacillin, erythromycin, clindamycin, ciprofloxacin, and moxifloxacin) and CC130 (harboring mecC; phenotypically resistant to penicillin, cefoxitin, and oxacillin only), respectively. . In 17 of the 66 non-single households, more than one person was concomitantly colonized. The observation of identical spa-types and resistance phenotypes in isolates from different individuals from the same household would initially indicate intrahousehold transmission. This was the case for 11 of these 17 households (Supplemental Table S1). Here, we observed 15 transmissions (calculated as the number of humans carrying S. aureus with matching typing characteristics minus one). Based on the 38 non-single households with at least one S. aureus carrier, where transmission could actually happen, the intrahousehold, human-to-human transmission rate was 11/38 (29.0%). The overall rate of S. aureus carriage among dogs was 10/112 (8.9%), i.e., dogs carrying S. aureus were found in 10/84 households (11.9%). However, of the ten dogs, eight lived in families where humans were also colonized with S. aureus. Among these eight households, there were three with two colonized persons. For all of these households, the isolates of the dogs exhibited the same spa-types and, with one exception, the same resistance phenotypes as those isolates derived from the humans living in the same household (Table 3), suggesting intrahousehold transmission between humans and dogs. This was confirmed by the results from the cgMLST allelic profiles derived from whole-genome sequencing of the 27 isolates ( Figure 1). Although we observed 1-15 allelic differences between the isolates, this still indicates very close relatedness and transmission [37]. As one transmission was likely for each of the eight households, the transmission rate was 8/42 (19%) and, thus, was lower than that observed for human-to-human transmission (11/38; p = 0.4306). A second sampling was performed for these eight households after one year, in which six households participated again. Besides a lack of detection of S. aureus in dogs from household No. 1 and, further, No. 2 and No. 4, the results from first sampling were confirmed (Table 3). Interestingly, in household No. 1, a second new dog (puppy) was acquired in that time period, and the human contact person living in that household lost the previous carriage of S. aureus CC15 and changed the nasal colonization to S. aureus CC45, closely related to that carried by the new dog (Table 3).

S. pseudintermedius Colonization of Dogs and Humans
This species was detected in nasal swabs from 42 (37.5%) of the 112 dogs who lived in 36 (42.8%) of the 84 households enrolled in the study. It was obtained from only 1 of the 179 humans (0.6%). Three of these dogs were concomitantly colonized with S. aureus. There was no association of S. pseudintermedius carriage and the dogs' hospital stays prior to sampling. Canine S. pseudintermedius carriage was not significantly associated with the number of dogs living in the same households (p 0.557, Table 4).    In only 1 of 22 households owning two dogs, S. pseudintermedius was detected in both dogs. These isolates were closely related when subjected to cgMLST, as shown in Figure 2 (cluster 1: two alleles different). In one of the three households owning three dogs, S. pseudintermedius was isolated from all three animals, and in one household owning four dogs, three carried S. pseudintermedius, respectively. In these cases, cgMLST typing revealed no relatedness. In only one household owning three dogs, S. pseudintermedius was detected in two of the three dogs and one of the three humans belonging to the household. The isolates from the human and from dog 1 were closely related (cluster 2; three alleles different), whereas the isolate from dog 2 was clearly not related. Altogether, the population structure of the sample of isolates investigated was highly diverse, as also indicated by attribution of the isolates to at least 12 complex types (Figure 2). The antibiotic resistance phenotypes of the 42 S. pseudintermedius isolates are shown in Table 5. Of these S. pseudintermedius isolates, 28 (68%) were susceptible to all tested antibiotics, 8 exhibited resistance to only one antibiotic, and 7 isolates were resistant to two or more antibiotics. One isolate (MRSP) was resistant to oxacillin (mecA positive), penicillin, erythromycin, clindamycin, tetracycline, and gentamicin. This isolate exhibited MLST ST672, as deduced from WGS data.

Cohort II: Dogs and Humans after Visiting a Veterinarian Practice
This group was composed of 17 pairs of dogs and their dedicated owners. Nasal S. aureus colonization was detected in 3 of the 17 humans and in 2 of the 17 dogs, which were not dedicated to the human carriers. S. pseudintermedius was obtained from six of the dogs, but none of the humans. Of these isolates, three were revealed as MRSP (Table 5).
S. pseudintermedius was detected in seven of the dogs (11.9%) and in one of the human owners (1.7%), whose dog did not carry S. pseudintermedius. The isolate from the human was multi-resistant (PEN, OXA, FOS, GEN, ERY, CLI, CIP, MOX, TRS, FUS) and mecApositive (MRSP).

Results from Spa-Typing and Phenotypic Susceptibility Testing of S. aureus (Cohorts I-III)
The results from spa-typing of the 87 isolates from humans are presented in supplemental Table S2. The attribution to clonal complexes of these isolates and of the 13 isolates from dogs is shown in Figure 3. Four clonal complexes predominate in this order: CC45 (n = 22), CC15 (n = 18), CC30 (n = 15), and CC8 (n = 13), demonstrated in Figure 3. The results from phenotypical antibiotic susceptibility testing of all detected isolates from the humans and dogs are compiled in supplemental Table S3.
The results from spa-typing of the 87 isolates from humans are presented in supplemental Table S2. The attribution to clonal complexes of these isolates and of the 13 isolates from dogs is shown in Figure 3. Four clonal complexes predominate in this order: CC45 (n = 22), CC15 (n = 18), CC30 (n = 15), and CC8 (n = 13), demonstrated in Figure 3. The results from phenotypical antibiotic susceptibility testing of all detected isolates from the humans and dogs are compiled in supplemental Table S3.

Discussion
S. aureus nasal carriage was observed in 36.9% of the humans living in 42 of 84 households owning dogs. This is more frequent than what has been observed in three previous studies in Central and Northern Germany (26.9% [38], 22% [39]), but in the range of data from other studies, indicating a prevalence of (intermittent) S. aureus carriage of 20-50% [2]. Among the factors that predispose a person for S. aureus carriage, such as diabetes mellitus or skin disorders/allergies, only atopic eczema was associated with S. aureus carriage in this study, which might have been due to the small sample size. The possible influence of living together with dogs on the nasal microbiome [40] and the consequences

Discussion
S. aureus nasal carriage was observed in 36.9% of the humans living in 42 of 84 households owning dogs. This is more frequent than what has been observed in three previous studies in Central and Northern Germany (26.9% [38], 22% [39]), but in the range of data from other studies, indicating a prevalence of (intermittent) S. aureus carriage of 20-50% [2]. Among the factors that predispose a person for S. aureus carriage, such as diabetes mellitus or skin disorders/allergies, only atopic eczema was associated with S. aureus carriage in this study, which might have been due to the small sample size. The possible influence of living together with dogs on the nasal microbiome [40] and the consequences on S. aureus colonization resulting therefrom remain to be explored. Among the 42 households with S. aureus carriage in humans, there were 17 in which two and more household members were colonized. Eleven of these isolates from different persons exhibited identical typing patterns, suggesting intrafamilial transmission between humans. This is well known and most probably mediated by direct contact and surface contamination in the household [41][42][43]. On the other hand, this means that, in 55.2% (21/38) of the non-single households with human carriers of S. aureus where one member was carrying S. aureus, the other human household members were not affected. We observed concomitant carriage of humans and dogs with closely related S. aureus isolates in eight households, which suggests transmission between humans and dogs (or vice versa). As it was observed in only 8 among the 42 households (19%) with human S. aureus carriers, it seems that transmission between humans and dogs occurs less frequently when compared to intrahousehold human-to-human transmission (39.529.0%, p=0.4306), although this effect was not statistically significant. Similar observations were reported from studies in Spain [44] and the USA [23,45]. Compared with our results, where the typing patterns of S. aureus from dogs and humans were identical and clearly indicated transmission, these studies partly found a higher proportion of different isolates. This might have been due to incomplete screening of all the humans in these households. The frequency distribution of clonal complexes to which the MSSA isolates from humans and dogs in our study were attributed corresponded to that observed in a previous study on nasal S. aureus colonization in Central Germany [36,39]. So far, there are no indications of dog-adapted clonal lineages of S. aureus. The results from a study on the dynamics of S. aureus colonization in humans and dogs in seven Spanish households [44] suggested humans as the original reservoir of S. aureus for dogs. Our results may support this hypothesis, as the overall carriage rates were much higher among humans than among dogs. However, transmission from dogs to humans also seems to be possible, as suggested by our observation that in one participant, a previously colonizing strain was displaced by another strain, which was also found in a dog that was bought shortly before the sampling. We detected MRSA in 2 of the 179 humans (1.1%), but not in the dogs. This prevalence in humans in the community corresponds to results from the previous studies mentioned above. One of these MRSA belonged to MLST clonal complex CC22, representing the majority of HA-MRSA isolates in Germany [46]. The other MRSA was associated with mecC and attributed to clonal lineage ST130, which has a wide range of animal hosts and is rare in humans so far [47]. Dogs acquire MRSA colonization by either animal hospital stay or by contact with humans with MRSA infection or colonization [26,28]; the canine carriage rates in German veterinary practices have previously been assessed and were 2.6%. This is higher than observed in this study. However, the sample size in the comparable cohort (cohort II) of this study was very small. We observed a lower prevalence of nasal S. aureus carriage in the humans handling therapy dogs (cohort III) vs. the humans living with dogs in their households (cohort I). An explanation could be that handlers of therapy dogs practice more careful hygiene, including hand washing, while at home or on duty with their dogs. Hands seem to play an important role in human-to-human transmission via the contamination of surfaces [43]. Another explanation may be that the contact is less intense. The prevalence of S. pseudintermedius carriage among dogs living in households was higher in our study (37.5%) than that reported for dogs from Spain (23%, [44]), but lower than observed in Canada (46%, [27]) and in Korea (66%, [16]). For the latter study, this could be due to the inclusion of rectal swabs, which might have increased the sensitivity of the analysis. In the study reported from Korea [16], the dogs were sampled at admission to veterinary hospitals. Interestingly, we observed no statistically significant association of the number of dogs living in the 36 households owning colonized dogs with the prevalence of nasal S. pseudintermedius colonization. This finding suggests that the acquisition of nasal colonization by transfer between healthy dogs probably does not frequently occur. This is in line with the observed highly diverse population structure, which was also reported for methicillin-susceptible S. pseudintermedius from France [48]. We observed nasal carriage of humans living with dogs in only 0.6% of the persons investigated. This is lower than reported from Korea [16], Spain [44], and Canada [27]. It seems that humans are not regular hosts for S. pseudintermedius. However, adaptation to humans may occur. A recent pilot study has shown diversity between the isolates attributed to the same clonal lineage from infections in humans and in dogs with respect to pathogenicity islands and virulence genecontaining prophages [49]. In this study, methicillin-resistant S. pseudintermedius (MRSP) was only detected in 0.9% of the dogs from households. This might have been due to the low number of dogs with a hospital stay prior to the study. The MRSP isolate exhibited MLST type ST610, which has not been reported so far for MRSP from dogs [12]. The prevalence of MRSP carriage by dogs in the community reported so far is 0.0% (Sweden, [14]), 2.6% (Norway, [50]), and 4.5% (Canada [27]). This is contrasted to the high prevalence observed in dogs undergoing clinical treatment [12,51]. Multi-resistant S. pseudintermedius seems to be associated with particular, animal-hospital-associated S. pseudintermedius clonal lineages [52], whereas isolates, which are susceptible or resistant to only a few antibiotics, represent natural colonizers of dog. Nevertheless, clinical microbiological laboratories should pay attention to the correct identification of S. pseudintermedius, which may be a more common human pathogen than has been recognized so far [53]. Although there is consensus that the benefits of AAI outweigh the risks with respect to the transmission of pathogens [29,54], the question of whether dogs used in AAI may be vectors of MSSA, MRSA, or MRSP remains of interest. Besides older observations [29,30], a current study in a pediatric hospital in the USA has shown mutual transmission of MRSA between dogs and children and the effectiveness of a decolonization procedure on dogs [55]. Guidelines for therapy animal organizations, facilities, and therapy animal handlers providing AAI in healthcare facilities were developed in several countries. SHEA (Society for Healthcare Epidemiology of America) has provided rather detailed guidance [56]. It recommends annual veterinary inspection of the dogs, but not routine screening for MRSA colonization. In contrast, the latter is recommended by the guideline from the German Society for Hospital Hygiene [57]. If the screening of dogs is required (e.g., for outbreak investigations), their handlers should also be screened, as mutual transmission cannot be excluded. So far, there is limited experience with the effectiveness of MRSA (or MSSA or MRSP) decolonization therapies for dogs, which may include the application of a mupirocin nasal ointment. In this context, the finding of high-level mupirocin resistance in MRSA from dogs must be taken into account [58,59]. With respect to alternatives, there is also a single report on the successful application of chlorhexidine [55].

Conclusions
We estimate the risk of acquisition of nasal colonization of humans with S. aureus from colonized dogs in households to be smaller than human-to-human transmission. For S. aureus, it seems to be higher than for S. pseudintermedius. Whether, in both species, clonal lineages with a more pronounced capacity for spreading among humans and dogs will emerge should be followed up through further surveillance.
Supplementary Materials: The following supplementary material is available online at https:// www.mdpi.com/article/10.3390/microorganisms10040677/s1. Table S1: Results from spa-typing of S. aureus isolates from humans and dogs; Table S2: Antibiotic resistance phenotypes of S. aureus from humans; Table S3: Antibiotic resistance phenotypes of S. aureus from humans.
Author Contributions: Conceptualization, C.C., W.W. and R.K.; recruitment of participants, C.C. and W.W.; primary diagnostics, C.C.; typing and input on analysis, F.L.-N. and R.W. data analysis, C.C.; writing-manuscript draft and feedback collection, C.C. All authors have read and agreed to the published version of the manuscript.
Funding: This study was partly performed within the framework of the German Research Network on Zoonotic Infectious Diseases (project #1 Health-PREVENT, grant no. 01KI2009F).
Data Availability Statement: Not applicable.